Cryopreservation of mouse and human oocytes using 1, 2-propanediol and the configuration of the meiotic spindle

Abstract

Human and mouse oocytes were cryopreserved by a slow freeze, rapid thaw method, using propanediol (PROH) as the cryoprotectant. A simulated cryopreservation was also included in the study to detect the level of damage attributable to the PROH alone. Comparison of the mouse and human oocytes cryopreserved by the same method showed opposing results, with a poor morphological survival rate of 4% observed for mouse oocytes and a subsequent normal fertilization rate of 0%. In 171 cryopreserved human oocytes a higher survival rate of 64% was achieved, and this showed more similarity to the mouse pronuclear oocytes survival of 53%. A comparison of human oocytes, cryopreserved within the cumulus and denuded of cumulus and corona prior to cryopreservation, demonstrated a higher survival rate in the denuded oocytes of 69% compared to 48%. A delay prior to cryopreservation of 1 or > or = 2 days had no effect on the immediate survival of oocytes, but culture for a further 24 h after thawing reduced survival, with the day 1 oocytes exhibiting the most dramatic reduction in survival (28%). Using a lectin binding method, abundant cortical granules were observed in all cryopreserved oocytes analysed. The meiotic spindle and chromosomes were examined in cryopreserved oocytes using fluorescence microscopy and 60% of the surviving oocytes had a normal spindle and chromosome configuration.

Qualitative and morphometric analysis of the ultrastructure of human oocytes cryopreserved by two alternative slow cooling protocols

“…The question of a nonphysiological discharge of CG in cryopreserved oocytes remains controversial. By using epifluorescence microscopy, Gook et al [37] showed that the staining specific for CG was not decreased in cryopreserved material in comparison to fresh controls. They also demonstrated that that fertilization could be achieved for frozen-thawed oocytes through standard IVF [28].…”

Live birth following transfer of a cryopreserved embryo generated from a cryopreserved oocyte and a cryopreserved sperm: Case report

“…Following rinsing in phosphate buffered saline (PBS) containing 20% heat-inactivated maternal serum, oocytes were dehydrated in solutions of the same basal medium containing 1.5 M 1,2 -propanediol (PROH) for 10 min followed by 1.5 M PROH + 0.2 M sucrose for a further 5 min before being loaded into plastic straws. Dehydration was performed at room temperature and cooling was as previously described [1]. Oocytes were frozen 7.5 h after follicle aspiration.…”

“…Oocyte thawing, fertilization and culture Straws containing cryopreserved oocytes were thawed rapidly as previously described [1] and rehydrated by sequential incubation (at 37 • C) in 1.0 M PROH/0.2 M sucrose (5 min), 0.5 M PROH/0.2 M sucrose (5 min), 0.2 M sucrose (2.5 min) and 0.1 M sucrose (2.5 min) before being washed twice in the basal medium and incubated in Quinn's Advantage Fertilisation medium containing 4 mg human serum albumin (HSA) per ml. The basal medium used for all rehydration solutions was Quinn's Advantage HEPES-buffered medium (QHEPES; SAGE BioPharma, USA) supplemented with 20 mg HSA per ml.…”

“…The resurgence of interest in human oocyte cryopreservation as a consequence of a number of reports suggesting that it may be a safe option in appropriate circumstances [1][2][3][4] has led to its clinical application and reports of a number of live births [5][6][7][8][9][10]. Although this approach has been adopted as an adjunct to routine IVF practice in Italy, this is predominantly a consequence of legal developments which resulted in the prohibition of the alternative, and more widepread, option of embryo cryopreservation.…”

Oocyte cryopreservation: is it time to remove its experimental label?

“…Early concerns regarding damage to the meiotic spindle [3], loss of cortical granules leading to lowered fertilization rates [4,5] and the low success rates of oocyte freezing/thawing as compared to the relative success of embryo cryopreservation caused a wavering of interest until the 1990s. Then, a series of studies indicating that reasonable oocyte thaw survival [6], fertilization [7,8], embryos with normal karyotype [8][9][10], and viable blastocyst development [11] could be achieved led to renewed interest in OC technology. Reports demonstrating live births following application of OC appeared soon after [12,13].…”